Novel Glu-based pyrazolo[3,4-d]pyrimidine analogues: design, synthesis and biological evaluation as DHFR and TS dual inhibitors

Abstract A novel series of multifunctional pyrazolo[3,4-d]pyrimidine-based glutamate analogs (6a–l and 7a,b) have been designed and synthesized as antifolate anticancer agents. Among the tested compounds, 6i exhibited the most potent anti-proliferative activity towards NSCLC, CNS, Ovarian, Prostate, Colon, Melanoma, Breast, and Renal cancers with good to weak cytostatic activity and non-lethal actions. 6i demonstrated higher selectivity for cancer than normal cells. 6i could significantly increase the accumulation of S-phase cells during the cell cycle distribution of cancer cells with high potency in the induction of apoptosis. The results unveiled that 6i probably acts through dual inhibition of DHFR and TS enzymes (IC50 = 2.41 and 8.88 µM, correspondingly). Docking studies of 6i displayed that N1-p-bromophenyl and C3-Methyl groups participate in substantial hydrophobic interactions. The drug-likeness features inferred that 6i met the acceptance criteria of Pfizer. Taking together, 6i could be a promising prototype for further optimization as an effective anticancer drug.


Introduction
During the process of DNA synthesis, a significant number of endogenic forces can challenge, and cells harbour a series of pathways to disseminate genome integrity 1 . Dihydrofolate reductase (DHFR) and thymidylate synthetase (TS) are key enzymes in nucleic acid synthesis, and they have long been evidenced as a crucial target of cancer chemotherapy 2,3 . DHFR reduces dihydrofolate (DHF) to tetrahydrofolate (THF) using NADPH, while TS catalyses the de novo synthesis of deoxythymidine monophosphate (dTMP) from deoxyuridine monophosphate (dUMP) using 5,10-Methylene THF as a cofactor, and thus initiates DNA synthesis and cell proliferation 2,3 . Therefore, DHFR and TS inhibitors cause THF and dTMP deficiency, resulting in RNA and DNA synthesis interference and apoptosis 4 . However, the dose-limiting toxicities, low solubility, short plasma half-life, low specificity, rapid diffusion throughout the body, weak brain permeability unless at high doses (1-8 g/m 2 ), low absorption and drug resistance development are the main drawbacks of the first-in-class DHFR and TS inhibitors [3][4][5] . A major mechanism of drug resistance by target cells to clinically useful classical antifolates ( Figure 1) is based on their need for polyglutamylation via the enzyme folylpoly-gamma-glutamate synthetase (FPGS), which catalyses the addition of several equivalents of glutamic acid to the c-carboxyl group in the side chain and hence prevents drug efflux from the cell 6 . Therefore, developing a novel antifolate class of low toxicity and tumour resistance remains one of the major challenges that incessantly attracts intense interest 2,3,5,7-10 .
The basic pharmacophoric features of classical antifolates such as methotrexate (MTX) 3 , pralatrexate (PDX) 3 , pemetrexed (PMX) 3 , and raltitrexed (RTX) 3 could be divided into four regions as shown in Figure 1: (1) the fused heterocyclic system (substituted pteridine, pyrrolo [2,3-d]pyrimidine and quinazoline scaffolds) which occupies the pteridine binding domain; (2) the heteroalkyl/alkyl chain (N-methyl aminomethyl, ethyl and sec-pentyne moieties) which occupies the bridge region and allows a tortuous "L" structure formation to fit tightly into the pocket of DHFR; (3) the benzoyl group which occupies the benzoyl region; and (4) the glutamate tail which occupies the l-glutamic acid binding site 3 . The SAR studies of classical antifolates usually vary in these regions.
The next generation of antifolates, as plevitrexed, revealed similar pharmacophoric properties of classical ones in which the free c-carboxylic group was replaced with bioisosteric tetrazole moiety which, in turn, either improved the drug absorption or did not need to be polyglutamylated by FPGS and hence allows more decrease in drug resistance. In this context, the nonclassical antifolates such as nolatrexed and piritrexim (PTX) were designed as they are more lipophilic and non-polyglutamatable agents 11,12 . Modifications in the antifolates structures have helped delineate the structural influence on the interaction with DHFR, TS, and FPGS utilisation. Therefore, intense interest has been in developing a new class of antifolate agents with these crucial structural pharmacophoric properties.
On the basis of isosterism and combination principles, as shown in Figure 1, the 6-5 fused system, pyrazolo [3,4-d]pyrimidine, was selected as pteridine core isostere since it has recently been reported to display potent anti-tumour activities against different cancer cells with multitarget properties [13][14][15][16] . The pyrazolo [3,4-d]pyrimidine core varied at N1, C3, and C6 and bridged at N5 with an acetamide linker which ended up with pharmacophoric benzoyl glutamate tail 3 . The linker acetamide was used to improve binding affinity through the formation of a tortuous "L" structure to fit tightly inside the active site. In the glutamate region, both a and c free carboxyl groups were blocked by making the corresponding ester in the form of a prodrug as a chemical approach catalysed by metabolic enzymes to minimise the undesirable physicochemical properties and enhance lipophilicity and obtain non-polyglutamatable agents 4,17 . Moreover, the fused pyrimidine scaffold was replaced by a pyrimidine core to study the effect of a 6-5 fused system on biological activity. Therefore, we aim to develop novel hDHFR and TS dual inhibitors as a new generation of novel effective multitarget antifolate prodrugs with fewer side effects. Herein, a new lipophilic Glu-based pyrazolo [3,4d]pyrimidine hybrid prodrug candidates were designed, prepared, and characterised by different spectroscopic techniques. The synthesised analogues were examined for their growth inhibitory (GI) effect on 59 cancer cell lines. The most active analogue was further subjected for its in vitro anti-proliferative activity against the most sensitive tumour and normal cell lines to investigate its selectivity towards cancer cells. In addition, cell cycle analysis and apoptosis were also examined. Finally, the in-silico molecular modelling and ADMET analysis were reported.
Moreover, as shown in Scheme 2, carboxylic acid derivatives 7a and 7b were prepared through alkaline hydrolysis of the corresponding diethyl ester analogues 6c and 6d, respectively. On the other hand, the Biginelli reaction was utilised to synthesise the 1,4-dihydropyrimidine derivatives 9a-c through S-alkylation of 2thioxo-1,4-dihydropyrimidine intermediates 8a-c with chloroacetamide glutamate derivative 5 as depicted in Scheme 3. Both spectral and elemental analysis data are depicted in the Supporting Information and have been fully consistent with the postulated structures of the prepared compounds.

Biological evaluation
Preliminary NCI-59 cancer cells single-dose screen The growth percentage (G%) and cell viability of the herein target compounds 6a-l, 7a,b, and 9a-c have been screened towards a full panel of 59 cancer cells at NCI's Developmental Therapeutics Program (NCI-DTP, MD, USA) 21 . The submitted analogues were initially examined for preliminary in vitro anticancer activity using sulforhodamine B (SRB) assay at 10 mM as a single dose to evaluate cell growth and viability according to the procedure of Drug Evaluation Branch 22,23 .
Different types of tumourscolon, NSCLC, leukaemia, CNS, renal, breast, ovarian, melanoma, and prostate cancerswere investigated as a full panel of 59 cell lines, and the attained results are cited in Figures S1-S17 (see Supplementary Material) where the value recorded is the growth relative to that of both the negative control and zero-time number of cells in which both growth inhibition (GI%; 0-100%) and lethality (L%; 0-100%) percentages can be identified. The growth inhibition means percentages (MGI%) for the examined target analogues towards the different treated 59 cancer cells were presented in Figure 2.
The attained results disclosed that compound 6k failed to demonstrate MGI%, while compounds 6a-h, 6j, 6 l, 7a,b, and 9a showed weak anticancer activity with MGI% ranging from 1.93% to 11.40%. Compounds 9b and 9c demonstrated moderate anticancer activity, with MGI% equal to 33.89% and 36.88%, respectively, while compound 6i exerted the significant mean growth inhibition percentage of 62.03% among the herein examined series. Superiorly, compound 6i emerged as the most efficient anticancer analogue eliciting high potency with broad-spectrum anticancer activity against all the herein subsets of cell panels, except against the A498 (Renal cancer) cell line ( Figure 2).
The inhibitory growth percentages (GI%) of compound 6i against the herein examined 59 cell panel are presented in Figure  3. Surprisingly, exploring the results hinted that compound 6i exhibited excellent anti-proliferative activities towards Breast

NCI 59 cancer cell five-dose screen
Remarkably, compound 6i (NSC: 838397) exhibited significant growth inhibition properties in the One-Dose (10 mM) screening assay; hence it was chosen for further evaluation at five-dose levels (0.01-100 mM) towards the herein examined tumour cell panels 21 (Figures S53-S56 in Supporting Information). This assay evaluates GI 50 , TGI, and LC 50 parameters, as cited in Table 1. Their values correspondingly represent the GI level, cytostatic impact, and cytotoxicity parameter. Furthermore, based on the GI 50 values, the mean graph midpoints (MG-MID) for both full NCI 59 cell panel and subpanel cancer cell lines were evaluated, affording a median potency parameter for the examined compound 6i, as depicted in Table 2.
Interestingly, the target compound 6i displayed potent anticancer activity at a single-digit micromolar concentration towards NSCLC ( (Table 1).
Furthermore, the target analogue 6i was found to be a nonlethal compound (LC 50 > 100 mM) towards most of the herein  (Table 1).
On the other hand, compound 6i had relatively homologous GI activity towards the cancer screening panel exploring the diverse cancer cells sensitivity, with efficient individual subpanel MG-MID values spanning from 6.63 to 68.03 mM and promising full panel MG-MID equals 20.45 mM. Interestingly, the most vulnerable subpanels to the impact of compound 6i were Non-Small Cell Lung and Prostate cancers with MG-MID equal 10.62 and 6.63 mM, correspondingly, Table 2.
Moreover, the selectivity index (SI) of the most efficient analogue 6i was calculated from the division of full-panel MG-MID by subpanel MG-MID. A value greater than 6 implies high selectivity, whereas a ratio from 3 to 6 denotes moderate selectivity. Otherwise, the compound is referred to as non-selective. 24 Hence, the examined compound 6i has emerged as a potent non-selective broad-spectrum anti-proliferative agent against all the herein examined subpanels with SI spanning in the interval: 0.3-1.9, except for Prostate cancer cell lines which stood out as a moderately selective agent with SI of 3.08, as depicted in Table 2. Therefore, the outstanding anti-cancer profile of compound 6i against the herein examined NCI-59 cell panels inspired us to explore its mechanism of action further.
Preliminary SAR study of the designed compounds As the tested compounds demonstrated, variable anti-proliferative potencies towards cancer cell lines and structure activity relationships (SAR) about these compounds could be summarised as follows. Firstly, the introduction of the methyl group at C6 of 6a,c,e,g,i and 7a (afforded 6b,d,f,h,j and 7b) would decrease their anti-proliferative effects while in compound 6k (afforded 6 l), the inhibitory activity  was increased by 8.18%. Secondly, the inhibitory activity of 6a,b, 6i, and 6l indicated that introducing substitution to the C3, especially methyl group, could significantly increase the inhibitory effects against the cancer cell lines. Thirdly, increasing lipophilicity at N1 by installing a p-bromophenyl substituent would significantly increase the inhibitory activity as in 6i. Fourthly, the inhibitory activity of 7a and 7b showed that hydrolysis of carboxylate ester of benzoyl glutamate tail of 6c and 6d would not improve the inhibitory effects. In turn, they could moderately decrease the inhibitory activities against most cancer cell lines evidencing that the ester prodrug form is more potent. Fifthly, replacing the pteridine isosteric 6-5 fused system, pyrazolo [3,4-d]pyrimidine, with 1,4-dihydropyrimidine core would significantly increase the inhibitory effect in compounds 9b and 9c. Also, the inhibitory effect of 9a, 9b, and 9c indicated that installing p-bromophenyl substituent at C4 in a pyrimidine ring could significantly increase the inhibitory effects against most of the cancer cells as in 6i, demonstrating the importance of this moiety on the anti-proliferative activity. Finally, 6i established better anti-proliferative effects among the tested analogues against NCI-59 cancer cells.
In brief, we could conclude that bridging of pyrazolo [3,4-d]pyrimidine core at C5 with diethyl glutamate tail through acetamide linker and installation of a more lipophilic group at N1 and a hydrophobic group at C3 along with no substitution at C6 could potentially impact the anti-proliferative activity ( Figure 4).

DHFR and TS inhibitory activity
To identify the effect on the targeted enzymes, hDHFR and TS inhibition assay was performed on the most potent anticancer analogue 6i. The assay was performed as reported elsewhere [32][33][34][35] using MTX and 5-FU as reference drugs, respectively. As shown in Table 4, the DHFR inhibition results established that 6i showed inhibitory potency of an IC 50 of 2.41 mM compared to MTX (IC 50 ¼ 0.11 mM). In contrast, the TS inhibition activity was comparable to that of 5-FU (IC 50 8.88 and 3.60 lM, respectively).

Cell cycle analysis in MCF-7 cells
The cell cycle distribution modulation and apoptosis induction were investigated to verify the mechanism of action of the most potent compound, 6i, in MCF-7 cells which were treated with 6i at 10 mM for 48 h along with an untreated DMSO control, washed with PBS, then fixed with 70% ice-cold ethanol for 12 h followed by staining with propidium iodide (PI), and analysed by flow cytometry 36,37 . The results showed that the percentage of S-phase cells increased to 36.29% for 6i from 24.95% (DMSO control) ( Figure 5(A-C)). These results indicated that 6i could effectively induce S-phase arrest.

Analysis of apoptosis
Again, MCF-7 cells were treated with 6i (10 mM) to assess its effect on cell apoptosis. As shown in Figure 5(D,E), the fraction of cells in early apoptosis were 26.27% and 0.43%, while in late apoptosis, they were 8.90% and 0.29% for compound 6i and DMSO control, respectively. The obtained results hinted that compound 6i could induce apoptotic pathways. However, the ratio of apoptosis inferred that the anti-proliferative effect of compound 6i was largely dependent on apoptosis.
In silico molecular modelling studies As seen in Figure 6(A,B), the docked pose of compound 6i (grey) showed an overlay with the cocrystallised ligand MTX (green) in the hDHFR active site 47 . The results display that the pyrazolo[3,4-d]pyrimidine core of 6i displayed only hydrophobic interactions: alkyl, pi-pi T-shaped, and pi-alkyl. In contrast, there are three hydrogen bonds for MTX in the corresponding binding site. The NH of the acetamide bridge showed a hydrogen bond with Asp21, while the benzoyl moiety demonstrated pi-alkyl hydrophobic interactions with Pro61:A and Pro26:A residues. The benzoylglutamate tail of 6i formed two hydrogen bonds from the c-carboxylate group with Asn64:A and Lys63:A residues, which differ from MTX 0 s. Also, compound 6i showed a FRED chemgauss4 score of À5.06, while MTX had a value of À13.38. The overlay of the docked pose of 6i with the crystal structure of MTX indicated that 6i established, to some extent, a similar binding mode with that of MTX, especially the formation of tortuous "L" structure, which allows tightly fitting in the active site of DHFR. This offered a plausible rationale for why 6i showed the best anti-cancer profile among the series tested, as established by NCI screening and molecular mechanisms. Furthermore, the molecular docking results infer that the hydrophobic interactions or hydrogen bonds of 6i were weaker than MTX 0 s. Consequently, 6i could be an inhibitor of DHFR but should be less potent than MTX. Overall, the Fred docking results proposed modifications or extensions on the N7 and benzoyl moiety as they projected towards the protein cavity.   48 . Like that of human DHFR, the TS active site consists of four regions: the pteridine, the bridge, the benzoyl, and the glutamate regions. The results display that the pyrazolo [3,4-d]pyrimidine core forms a hydrogen bond between bromine and Arg50:A and hydrophobic interactions: pi-pi Tshaped, alkyl, and pi-alkyl. The acetamide linker allows an overlay with PMX crystal structure through tortuous "L" structure formation. The benzoyl moiety formed mixed pi-alkyl hydrophobic interactions with Leu221:A. Finally, the glutamate tail of 6i forms van der Waals attractions in the benzoylglutamate region. The binding pose of 6i indicates that the pyrazolo [3,4-d]pyrimidine motif and benzoylglutamate tail form a tortuous "L" structure through the acetamide bridge region and spatially stacked well with the docked pose of PMX. Furthermore, the modelling study implies that 6i could be an inhibitor of TS but should be less potent than PMX, which is aligned with the results of inhibitory enzyme assays. Overall, the Fred docking results proposed modifications or extensions on the N1, N7, benzoyl moiety, and c-carboxylic ester group as they project towards the protein cavity.

Drug-likeness and ADMET prediction
Drug-likeness and pharmacokinetics ADMET properties for the most active compound 6i were calculated using ADMETlab 2.0 predictor 49,50 . As illustrated in Table 5, Only one violation from Lipinski's rule of five is permitted for a compound to obey the rule. It can be observed that compound 6i revealed two violations from Lipinski's rule while Pfizer accepts 6i criteria as a good prodrug candidate. The ADMET prediction data of compound 6i are cited in Table 6. The results revealed that 6i has a good synthetic accessibility score (SA score 3.14), a high GIT absorption rate since the human intestinal absorption (HIA) is 90% or more, and moderate solubility in water that helps in solubilisation in GIT fluids before absorption process as indicated by the estimated log S value (À5.07). In addition, the tested compound 6i demonstrated more than 20% bioavailability, as indicated by the F 20% result, while the plasma protein binding was 91.72%. Also, it is expected to be a non-inhibitor of CYP1A2, CYP2C19, and CYP2D6 enzymes, suggesting a low probability of causing drug-drug interaction. Furthermore, carcinogenicity and acute toxicity tests showed that compound 6i is neither carcinogenic nor toxic, evidencing the good ADMET profile.

Experimental part
Chemistry Bruker Avance III 400 MHz HD FT-high resolution-NMR spectrometer was used for recording the spectra of 1 H and 13 C NMR in deuterated solvents DMSO-d 6 and CDCl 3 using the residual peaks at dH ¼ 2.50 ppm and dH ¼ 7.27 ppm as an internal standard for 1 H NMR  spectra. In contrast, dC ¼ 39.51 ppm and dC ¼ 77.23 ppm were used for 13 C NMR spectra, respectively. The multiplicity of signals was verified as singlet (s), doublet (d), doublet of doublets (dd), triplet (t), quartette (q), multiplet (m), or broad (br). The J-coupling constants (in Hz) were recorded. For reaction monitoring, silica gel 60 F 254 pre-coated plates (Merck) were used for TLC using UV light (254 nm) for visualisation. The purification processes of the prepared analogues were accomplished by either recrystallisation from an appropriate solvent or chromatographic separation utilising silica gel for column (150-250 lm). All melting points were determined on an electro thermal melting point apparatus (Stuart Scientific, Model SMP1, UK) and were uncorrected. Mass spectrum was carried out on Direct Inlet part to mass analyser in Thermo Scientific GC-MS model ISQ (Waltham, MA, USA). Vario elemental analyser III (Vario, Germany) was used for analysing C, H, and N for all designed analogues. The findings agreed with the proposed structures within ±0.4% of the theoretical values. Dry solvents and chemicals were purchased from commercial suppliers and used as received. Compounds 2a-f 18,19,51-53 , 3a-e 18,19,51,54-58 , 3gl 18,19,51,54-58 , 5 59 and 8a-c 60-64 were synthesised as described.
General procedure for synthesis of compounds 3a-l To a suspension of 1-aryl-5-amino-4-cyanopyrazoles 2a-f (0.006 mol) in aliphatic acid (18 ml), POCl 3 (1.2 ml) was added instantaneously. The mixture was refluxed for 2 h 20 and then cooled to 0 C, poured slowly to crushed ice, filtered, and recrystallised from formic acid (85%) to provide the intermediates 3a-l in good yields.
General procedure for the synthesis of acid derivatives (7a,b) A suspension of 6c,d (0.002 mol) in 5 ml aqueous sodium hydroxide (1 mol/l) was stirred at 25 C for 2 h (TLC monitoring) 3 . The reaction mixture was then filtered and acidified carefully with 0.5 mol/l HCl. The formed product was filtered and crystallised from the appropriate solvent to give 7a,b.
The key intermediate (3f) and target compounds (6a-l, 7a,b, and 9a-c) were fully characterised in the Supplementary Material.

NCI-60 screening methodology
The designed analogues were submitted to NCI-Chemotherapeutic Agents Repository (MD, USA) for screening on a panel of 59 cancer cell lines. All compounds were initially examined at 10 mM concentration (Single-Dose Screen), and then the most efficient analogue 6i was screened further at 0.01-100 mM (Five-Dose Screen). The screening procedures were performed according to NCI-Drug Evaluation Branch protocol using SRB protein assay, as described previously 21,66 . The experimental procedures were detailed in the Supporting Information.

SI ¼ IC 50 HSF cells IC 50 of cancer cells
Cell cycle analysis The cell cycle assay was carried out according to the previously reported 36,37 . Briefly, in a six-well plate, 2 ml of MCF-7 cells were   seeded per well and incubated overnight at 37 C and CO 2 (5%). After 12 h incubation with serum-free medium, they were treated with 10 lM of 6i, incubated for 48 h, then washed twice with 1Â PBS, trypsinised and centrifuged for 5 min (500 xg) at 4 C. And then the cell pellet, in ice-cold 1Â PBS, was fixed with absolute ethanol at À20 C for 2 h, washed with 1Â PBS ice-cold solution and finally stained with propidium iodide (PI)/RNase (Abcam, ab139418) for 0.5 h at 25 C in the dark. flow cytometry determined the DNA content in cell phases using BD FACS Calibur 36,37 .

Apoptosis analysis
The MCF-7 cells (10 6 cells/ml) were treated with either 10 mM of 6i or DMSO (NC) for 24 h. After that, the cells were trypsinised, collected, washed, and stained with PE-Annexin-V (BioVision, K101-25) in the presence of PI 67 . The induction of apoptosis was analysed by flow cytometry using BD FACS Calibur 67 .
DHFR inhibitory assay DHFR inhibition assay kit (BioVision, K247-100) supplied with recombinant hDHFR was utilised in this assay. The assay was performed as instructed in assay kit 32 . Briefly, in a 96-well clear plate, serial dilutions of the test compound (2 lL) were loaded. DHFR was diluted in assay buffer (400-fold), and 98 lL was added to the assigned wells for compound 6i and enzyme control. An assay buffer (100 ml) was used as a Background Control. Then, 40 lL of diluted NADPH in assay buffer (40-fold dilution) was added and mixed well. At 25 C in the dark, the plate was incubated for 15 min. After that, the diluted substrate in assay buffer at 15-fold dilution (60 lL) was added to assigned wells. Instantaneously, the absorbance was read at 340 nm for 10-20 min at 25 C in kinetic mode by Robonik ELISA plate reader. Finally, the activity and relative inhibition percentages were measured using the equations indicated in the assay kit [33][34][35] .

Drug-likeness and ADMET analyses
Drug-likeness, physicochemical and pharmacokinetic characters of the most potent analogue 6i were predicted using an accurate, comprehensive, and efficient web-based tool ADMETlab 2.0. They were utilising a high-quality database and a framework for multitask graph attention 49,50 .

Molecular modelling studies
The straightforward OpenEyeV R scientific software (2021.2.1) was used for molecular docking studies 19,[38][39][40][41][42] . A library of the most potent compound 6i, MTX, and PMX was built, and MMFF94 force field was used for energy minimisation then generation of multiconformers was offered by OMEGA V R module 68 . Both hDHFR (PDB ID 1U72) and TS (PDB ID 1JU6) receptors were prepared by the Spruce V R application, and then FRED V R module was utilised for molecular modelling, and hence FRED Chemgauss4 score generation 69 . The Vida V R module was used to visualise the tested compounds 0 poses to both DHFR and TS active sites 70 . Also, a 2D diagram of binding interactions was generated by the Discovery Studio Visualisation tool (DS) 71,72 . The Docking-Report module generated the docking report through the command lines stated in OpenEye documentation 73 .

Conclusion
In this research, a novel series of multifunctional pyrazolo [3,4d]pyrimidine and 1,4-dihydropyrimidine Glu-based derivatives 6al, 7a,b, and 9a-c were designed and synthesised as potential antifolate agents. The NCI-59 Screening assay displayed that compounds 6i and 9a-c showed the best MGI% among the series investigated. At the NCI five-dose assay, compound 6i exhibited potent anti-proliferative activity against the herein tumour screening panel with GI 50 spanning in the range: 3.93-9.85 mM.
Compound 6i revealed good to weak cytostatic activity with TGI spanning in the range: 16.50-89.10 mM, and it was found to be a non-lethal compound (LC 50 > 100 mM) towards most of the herein examined cancer cell lines. Moreover, compound 6i elicited broadspectrum anticancer activity with effective subpanel MG-MID interval: 6.63-68.03 mM and promising full panel MG-MID of 20.45 mM with moderate selectivity towards Prostate cancer cell lines. The target analogue 6i, as the most potent derivative, demonstrated high selectivity towards cancer cell lines with IC 50 values equal to 4.40, 12.42, and 11.50 against MCF-7, PC-3, and OVCAR-3 cells, respectively. Furthermore, compound 6i showed potent hDHFR inhibitory activity with an IC 50 equal to 2.41 mM compared to MTX (IC 50 ¼ 0.11 mM), while the inhibitory activity towards TS was 8.88 mM compared to 5-FU (IC 50 ¼ 3.6 mM). The target compound 6i probably acts through dual inhibition of DHFR and TS enzymes. Compound 6i could significantly arrest the cells at the S-phase of the cell cycle and potentially induce apoptosis. The in silico molecular docking studies implied that compound 6i had an overlay with the crystal structure of MTX and PMX in their active sites of DHFR and TS, respectively, forming a tortuous "L" structure through the acetamide bridge region. The drug-likeness and ADMET analysis revealed that 6i met the Pfizer acceptance criteria. These results thoroughly delineate 6i as a good lead compound and multi-targeted antifolate agent for further study.